Contrast agents for magnetic resonance imaging and methods related thereto

ABSTRACT

In certain aspects the present invention provides methods and compositions related to contrast agents for magnetic resonance imaging. In certain variations, contrast agents provided herein are generated in situ via genetic instructions and become potent upon sequestering available metal atoms. Exemplary contrast agents include metal-binding proteins.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 60/363,163, filed on Mar. 7, 2002, whichapplication is hereby incorporated by reference in its entirety.

INTRODUCTION

Tools that enable one to visualize gene expression in vivo are offundamental importance to the future of medicine and the biologicalsciences. The emerging field of genetic medicine requires non-invasiveimaging methods that can indicate where, when and if therapeutic geneshave been delivered and whether the desired protein has been expressed.In the realm of basic biological research, the ability to image thetiming and location of gene expression in vivo is a fundamental need.

Scientists typically monitor gene expression by incorporating a markergene that is expressed along with the gene of interest, often as eithera transcriptional or translational fusion. Detection of the marker geneproducts is most often achieved using histological preparations (e.g.using a β-galactosidase assay), or by using fluorescence microscopy(e.g. using green fluorescent protein, or GFP). Neither of these methodspermit non-invasive imaging of tissues or other macroscopic assembliesof cells. Markers that require histological preparation cannot bedetected without sacrificing the subject material. Fluorescent markerscan be imaged in living cells, but even with the most sophisticatedoptical technologies available, it is not possible to image at tissuedepths exceeding approximately 500 μm. Other methods such as PET(positron emission tomography), gamma cameras, and SPECT (single-photonemission computed tomography) have been used to detect gene expressionin vivo, but all of these suffer from limited spatial resolution, whichis on the order of cubic millimeters or larger.

MRI is a widely used clinical diagnostic tool that allows non-invasiveimaging of optically opaque subjects and provides contrast among softtissues at high spatial resolution. In the majority of clinicalapplications, the MRI signal is derived from protons of the watermolecules present in the materials being imaged. The image intensity oftissues is determined by a number of factors. The physical properties ofa specific tissue, such as the proton density, spin lattice relaxationtime (T1), and the spin-spin relaxation time (T2) often determine theamount of signal available.

A number of compositions termed “contrast agents” have been developed toprovide enhanced contrast between different tissues. Contrast agentscommonly affect T1, T2 or both. In general, contrast agents are madepotent by incorporating metals with unpaired d or f electrons. Forexample, T1 contrast agents often include a lanthanide metal ion,usually Gd³⁺, that is chelated to a low molecular-weight molecule inorder to limit toxicity. T2-agents often consist of small particles ofmagnetite (FeO—Fe₂O₃) that are coated with dextran. Both types of agentsinteract with mobile water in tissue to produce contrast; the details ofthis microscopic interaction differ depending on the agent type.

Most widely used contrast agents are exogenous, meaning that thecontrast agent is produced externally and then delivered to the tissueor cells to be imaged. Exogenous contrast agents are generally deliveredthrough the vascular system, typically have a nonselective distribution,and are physiologically inert. The exogenous contrast agents are used tohighlight anatomy with poor intrinsic contrast, as well as to visualizevarious pathologies that disrupt normal vascular flow or cause a breakin the blood-brain-barrier. None of these agents cross cellularmembranes easily and therefore the existing technology is difficult toadapt for the analysis of intracellular events.

A new generation of MRI contrast agents is required to adapt thispowerful imaging technology to the needs of molecular medicine andbiological research.

SUMMARY OF THE INVENTION

In certain aspects, the invention relates to contrast agents formagnetic resonance imaging that are synthesized in a subject material asdirected by a nucleic acid sequence. The contrast agents are made potentby sequestering available metal atoms, typically iron atoms. In certainaspects, the nucleic acid sequence encodes a metal binding protein thatacts, directly or indirectly, to impart a contrast effect on the cell inwhich it is produced. The invention further relates to methods ofgenerating and employing the subject contrast agents.

In certain embodiments, the invention relates to methods of generatingan image of a subject material by imaging a subject material comprisinga plurality of cells wherein a subset of the cells contain anMRI-detectable amount of contrast protein. In preferred embodiments, theamount of contrast protein present in different cells isdistinguishable, and optionally, cells comprising measurable amounts ofcontrast protein are distinguishable from cells or other components ofthe material that do not comprise the measurable amount of contrastprotein.

In another embodiment, methods of the invention comprise detecting geneexpression by imaging a cell comprising a recombinant nucleic acidencoding a contrast agent. Preferably, detection of the contrast proteinby magnetic resonance imaging indicates that the nucleic acid encodingthe contrast protein is and/or has been expressed. Optionally, thecontrast agent is a protein, preferably a metal-binding protein.Exemplary classes of metal binding proteins include ferritin proteins;transferrin receptor proteins; iron regulatory proteins; and ironscavenger proteins. Exemplary metal binding proteins of the inventioninclude metal binding proteins that are at least 60%, optionally atleast 70%, 80%, 90%, 95%, 99% or 100% identical to a sequence as shownin any of SEQ ID Nos: 2, 4, 6, 8, 10, 12, and 14. Alternatively, theprotein is at least 60%, optionally at least 70%, 80%, 90%, 95%, 99% or100% identical to a sequence as shown in any of SEQ ID Nos: 16, 18, 20or 22.

Methods described herein may be used with essentially any materialcapable of generating the contrast agent in situ. For example, thesubject material may be a cell, optionally a cell that is part of a cellculture, part of an in vitro tissue or part of a multicellular organism,such as, for example, a fungus, a plant, or an animal. In preferredembodiments, the subject material is a living mammal such as a mouse ora human.

In further aspects, the invention provides vectors for transfection of amulticellular organism comprising a recombinant nucleic acid encoding acontrast agent. In certain embodiments, the contrast agent is ametal-binding protein. Optionally, the vector is a viral vector derivedfrom a virus selected from the group: an adenovirus, anadenovirus-associated virus, a herpes simplex virus, a retrovirus, analphavirus, a poxvirus, an arena virus, a vaccinia virus, an influenzavirus, a polio virus and a hybrid of any of the foregoing.

In additional aspects, the invention includes delivery systems forintroducing nucleic acids of the invention into subject material. Incertain embodiments, the invention provides viral particles suitable fortransfecting a mammalian cell, comprising a nucleic acid comprising acoding sequence for a contrast agent, such as a contrast agent describedabove. Optionally, the viral particle is derived from one or more of thefollowing: an adenovirus, an adenovirus-associated virus, a herpessimplex virus, a retrovirus, an alphavirus, a poxvirus, an arena virus,a vaccinia virus, an influenza virus and a polio virus. In additionalembodiments, the invention provides colloidal suspensions suitable fortransfecting a mammalian cell comprising a nucleic acid comprising acoding sequence for a contrast agent, such as a contrast agent describedabove. Optional types of colloidal suspensions include one or more ofthe following: a macromolecule complex, a nanocapsule, a microsphere, abead, an oil-in-water emulsions, a micelle, a mixed micelle, and aliposomes.

In yet further aspects, the invention provides cells, cell cultures,organized cell cultures, tissues, organs and non-human organismscomprising a recombinant nucleic acid comprising a coding sequence for acontrast agent, such as a contrast agent described above. In certainembodiments, the organism is selected from the group consisting of: amouse, a rat, a dog, a monkey, a pig, a fruit fly, a nematode worm and afish, or alternatively a plant or fungus. In further embodiments, thecells, cell cultures, organized cell cultures, tissues, organs andnon-human organisms may comprise a vector as described above.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

The claims provided below are hereby incorporated into this section byreference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Correlation between ferritin increase and 1/T₁ (a) and 1/T₂ (b)in simulated tumors. The solid line is least-squares fit through thedata (guide for the eye). The values are normalized to give the ferritinincrease over the mean baseline value of the control pellet, which is1.5 mg/ml of ferritin; the experimental samples were incubated withvarious concentration of ferric ammonium citrate (FAC) and the controlsamples were incubated in the absence of FAC. The error bars representthe standard deviation for N=4 experimental runs.

FIG. 2. Data showing the percent of the total number cells remainingafter the 16 hour period of ferritin loading. For each FAC concentration(and control), cells before and after the incubation period were counted3-times using a hemocytometer and the results were averaged. The errorbars represent the standard deviation for the separate (N=4) incubationexperiments.

FIG. 3. MRI image of three simulated tumor samples. Here, (a) is thecontrol and (b) and (c) are the samples containing a ferritin increaseof 2.7 and 4, respectively. Contrast among these samples is readilyapparent in this T₂-weighted image. Images were acquired simultaneouslyusing a Bruker 7-Tesla MRI system with TE/TR=45/2000 ms, 128×128 imagepoints, and a 1 mm-thick slice. The pellet size was approximately 4 mmin diameter.

FIG. 4: MRI image through pelleted 9L glioma cells transfected withcontrast proteins light (LF) and heavy (HF) chain ferritin. The sampleon the left is the control (no DNA added during incubation). Imagecontrast is readily apparent between the two pellets. Expression of thereporter turns cells dark in the MR image. This image was acquired usingan 11.7 Tesla MRI system with a standard T₂-weighted 2DFT pulsesequence. This image was acquired at 4° C.

FIG. 5: MRI image through pelleted 9L cells infected with contrastproteins light (LF) and heavy (HF) chain ferritin via an adenovirus. Thesample on the left is the control (uninfected cells). Image contrast isreadily apparent between the two pellets. (Note that the intense darkspots in the pellets are bubble artifacts.) This image was acquiredusing an 11.7 Tesla MRI system and a standard T₂-weighted 2DFT pulsesequence. This image was acquired at 4° C.

FIG. 6: Human ferritin heavy chain cDNA sequence (BC016009) (SEQ IDNO:1). The coding region is underlined.

FIG. 7: Human ferritin light chain cDNA sequence (XM_(—)050469) (SEQ IDNO:3) The coding region is underlined.

FIG. 8: Mus musculus ferritin heavy chain cDNA sequence (NM_(—)010239.1)(SEQ ID NO:5). The coding region is underlined.

FIG. 9: Mus musculus ferritin light chain 1 cDNA sequence(NM_(—)010240.1) (SEQ ID NO:7). The coding region is underlined.

FIG. 10: Mus musculus ferritin light chain 2 cDNA sequence(NM_(—)008049.1) (SEQ ID NO:9). The coding region is underlined.

FIG. 11: Rattus norvegicus ferritin subunit H cDNA sequence(NM_(—)012848.1) (SEQ ID NO:11). The coding region is underlined.

FIG. 12: Homo sapiens transferrin receptor cDNA sequence (NM_(—)003234)(SEQ ID NO:15). The coding region is underlined.

FIG. 13: Homo sapiens transferrin receptor 2 cDNA sequence(NM_(—)003227) (SEQ ID NO:17). The coding region is underlined.

FIG. 14: Mus musculus transferrin receptor 2 nucleic acid sequence(NM_(—)015799) (SEQ ID NO:21). The coding region is underlined.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

“Coding sequence” is used herein to refer to the portion of a nucleicacid that encodes a particular protein. A coding region may beinterrupted by introns and other non-coding sequences that areultimately removed prior to translation.

“Colloidal suspension” is used herein to refer to a colloidal suspensionthat comprises one or more nucleic acids for delivery to cells. Thematerial in a colloidal suspension is generally designed so as toprotect nucleic acids and facilitate the delivery of nucleic acidsacross cell membranes. Exemplary colloidal suspensions include, but arenot limited to, lipid micelles, tubes, rafts, sandwiches and other lipidstructures, often comprising cationic lipids. Other colloidalsuspensions include nanocapsules, microbeads and small, nucleicacid-binding polymeric structures, etc.

The term “contrast agent” is used herein to refer to a molecule thatgenerates a contrasting effect in vivo, whether the effect is direct orindirect or both. In exemplary embodiments, “contrast agent” is usedinterchangeably with “contrast protein” or “contrast polypeptide.” Inthe case of a direct effector, the contrast protein will typically forma complex that affects the relaxation times T1, T2 or T2*. Often directcontrast proteins form metalloprotein complexes. Exemplary categories ofcontrast proteins include, for example, metal binding proteins and/oragents that stimulate production of one or more metal-binding protein,etc. Indirect effectors include molecules that cause a cell to produce adirect contrast protein and/or modulate a functional, biochemical,and/or biophysical characteristic of a direct contrast protein, therebycreating a contrast effect. Exemplary categories of indirect effectorsinclude, for example, proteins and/or nucleic acids that affectexpression of a direct contrast protein, modulate the activity of adirect contrast protein, modulate metal binding to a metal-bindingprotein, modulate expression of an iron regulatory protein, and/ormodulate the activity of an iron regulatory protein, etc.

The term “contrast effect”, as used herein with respect to MRI, includesany alteration in the MRI signal that renders one cell or tissuedetectably different from another. A contrast effect may involve effectson T1, T2 and/or T2*. In MRI, a subject containing mobile water isgenerally placed in a large static magnetic field. The field tends toalign some of the magnetic moments (spins) of the hydrogen nuclei in thewater along the field direction. The spin lattice relaxation time (T1)is the time constant for a population of nuclei placed in a magneticfield to equilibrate along the magnetic field direction. T1 is the timeconstant for the transfer of energy from the spin system to theenvironment (the lattice). The spin-spin relaxation time (T2) is thetime constant for nuclei precessing at the Larmor frequency to remain inphase with each other. Alternatively, T2 is called the spin-phase memorytime. This loss of phase coherence is attributed to low-frequencyfluctuations of the magnetic field that are commonly due to interactionsamong spins. The relaxation time T2* is defined as 1/T2*=1/T2+γΔB, whereγ is the nuclear gyromagnetic ratio and ΔB is the static externalmagnetic field inhomogeneity.

The terms “contrast gene” or “contrast nucleic acid” are usedinterchangeably herein to refer to a nucleic acid comprising a codingsequence for a contrast protein.

An “externally regulated promoter” is a nucleic acid that affectstranscription in response to conditions that may be provided in acontrolled manner by one of skill in the art. Externally regulatedpromoters may be regulated by specific chemicals, such as tetracyclineor IPTG, or by other conditions such as temperature, pH, oxidation stateetc. that are readily controlled external to the site of transcription.

The term “Ferritin protein” is intended to include any of a group ofdiiron-carboxylate proteins characterized by the tendency to form amultimeric structure with bound iron and having a helix-bundle structurecomprising an iron-coordinating Glu residue in a first helix and aGlu-X-X-His motif in a second. Certain ferritins maintain bound iron ina primarily Fe(III) state. Bacterioferritins tend to be haem proteins.Vertebrate ferritins tend to be assembled from two or more subunits, andmammalian ferritins are often assembled from a heavy chain and a lightchain. Many ferritins form hollow structures with an iron-rich aggregatein the interior. Exemplary ferritins are presented in Table 1 below.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two polypeptides or between two nucleic acid molecules. Homologyand identity can each be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. When anequivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position;when the equivalent site occupied by the same or a similar amino acidresidue (e.g., similar in steric and/or electronic nature), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of identical or similar amino acids atpositions shared by the compared sequences. A sequence which is“unrelated” or “non-homologous” shares less than 40% identity, thoughpreferably less than 25% identity with a sequence of the presentinvention.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention may be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences or homologs. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.(1990) J Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used. See http://www.ncbi.nlm.nih.gov.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990).

The term “iron binding protein” as used herein is intended to includeproteins that bind to iron under physiologically relevant conditions.Certain iron binding proteins interact with iron through a cofactor suchas heme. Many other exemplary cofactors are also described herein. Otheriron binding proteins form an iron binding site with the appropriateamino acids, including but not limited to, histidine, aspartate,glutamate, asparagine and glutamine. Although iron binding proteins ofthe invention bind iron, they are also likely to bind to other metals.Accordingly, “iron binding protein” as used herein is not meant toindicate that the protein binds iron exclusively, or even that theprotein binds iron more tightly than other metals.

An “iron regulatory protein” refers to a protein that is involved iniron utilization, processing, and/or accumulation in a cell. Ironregulatory proteins include, for example, proteins that regulate ironhomeostasis, proteins that regulate iron trafficking into or out of acell, proteins involved in regulating the production of iron relatedelements, such as, for example, ferritin and transferring, etc. Ironregulatory proteins may or may not bind iron directly.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include analogs of eitherRNA or DNA made from nucleotide analogs (including analogs with respectto the base and/or the backbone, for example, peptide nucleic acids,locked nucleic acids, mannitol nucleic acids etc.), and, as applicableto the embodiment being described, single-stranded (such as sense orantisense), double-stranded or higher order polynucleotides.

The term “operably linked” is used herein to refer to the relationshipbetween a regulatory sequence and a gene. If the regulatory sequence ispositioned relative to the gene such that the regulatory sequence isable to exert a measurable effect on the amount of gene productproduced, then the regulatory sequence is operably linked to the gene.

A “polylinker” is a nucleic acid comprising at least two, and preferablythree, four or more restriction sites for cleavage by one or morerestriction enzymes. The restriction sites may be overlapping. Eachrestriction sites is preferably five, six, seven, eight or morenucleotides in length.

A “recombinant helper nucleic acid” or more simply “helper nucleic acid”is a nucleic acid which encodes functional components that allow asecond nucleic acid to be encapsidated in a capsid. Typically, in thecontext of the present invention, the helper plasmid, or other nucleicacid, encodes viral functions and structural proteins which allow arecombinant viral vector to be encapsidated into a capsid. In onepreferred embodiment, a recombinant adeno-associated virus (AAV) helpernucleic acid is a plasmid encoding AAV polypeptides, and lacking the AAVITR regions. For example, in one embodiment, the helper plasmid encodesthe AAV genome, with the exception of the AAV ITR regions, which arereplaced with adenovirus ITR sequences. This permits replication andencapsidation of the AAV replication defective recombinant vector, whilepreventing generation of wild-type AAV virus, e.g., by recombination.

A “regulatory nucleic acid” or “regulatory sequence” includes anynucleic acid that can exert an effect on the transcription of anoperably linked open reading frame. A regulatory nucleic acid may be acore promoter, an enhancer or repressor element, a completetranscriptional regulatory region or a functional portion of any of thepreceding. Mutant versions of the preceding may also be consideredregulatory nucleic acids.

A “transcriptional fusion” is a nucleic acid construct that causes theexpression of an mRNA comprising at least two coding regions. In otherwords, two or more open reading frames may be organized into atranscriptional fusion such that both open reading frames will beexpressed as part of a single mRNA and then give rise, as specified bythe host cell, to separate polypeptides. The open reading frames in atranscriptional fusion tend to be subject to the same transcriptionalregulation, but the encoded polypeptides may be subject to distinctpost-translational fates (eg. degradation, etc.). A “transcriptionalfusion” may be contrasted with a “translational fusion” in which two ormore open reading frames are connected so as to give rise to a singlepolypeptide. The fused polypeptides in a “translational fusion” tend toexperience similar transcriptional, translational and post-translationalregulation.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell, and isintended to include commonly used terms such as “infect” with respect toa virus or viral vector. The term “transduction” is generally usedherein when the transfection with a nucleic acid is by viral delivery ofthe nucleic acid. “Transformation”, as used herein, refers to a processin which a cell's genotype is changed as a result of the cellular uptakeof exogenous DNA or RNA, and, for example, the transformed cellexpresses a recombinant form of a polypeptide or, in the case ofanti-sense expression from the transferred gene, the expression of anaturally-occurring form of the recombinant protein is disrupted.

As used herein, the term “transgene” refers to a nucleic acid sequencewhich has been introduced into a cell. Daughter cells deriving from acell in which a transgene has been introduced are also said to containthe transgene (unless it has been deleted). A transgene can encode,e.g., a polypeptide, partly or entirely heterologous, i.e., foreign, tothe transgenic animal or cell into which it is introduced. Optionally, atransgene-encoded polypeptide may be homologous to an endogenous gene ofthe transgenic animal or cell into which it is introduced, but may bedesigned to be inserted, or is inserted, into the genome in such a wayas to alter the genome of the cell into which it is inserted (e.g., itis inserted at a location which differs from that of the natural gene).Alternatively, a transgene can also be present in an episome. Atransgene can include one or more transcriptional regulatory sequencesand any other nucleic acid, (e.g. intron), that may be necessary foroptimal expression of a selected coding sequence. A transgene may alsocontain no polypeptide coding region, but in such cases will generallydirect expression of a functionally active RNA, such as an rRNA, tRNA,ribozyme, etc. A “therapeutic transgene” is a transgene that isintroduced into a cell, tissue and/or organism for the purpose ofaltering a biological function in a manner that is beneficial to asubject.

“Transient transfection” refers to cases where exogenous nucleic acid isretained for a relatively short period of time, often when the nucleicacid does not integrate into the genome of a transfected cell, e.g.,where episomal DNA is transcribed into mRNA and translated into protein.A cell has been “stably transfected” with a nucleic acid constructcomprising viral coding regions when the nucleic acid construct has beenintroduced inside the cell membrane and the viral coding regions arecapable of being inherited by daughter cells.

“Viral particle” is an assemblage of at least one nucleic acid and acoat comprising at least one viral protein. In general, viral particlesfor use in delivering nucleic acids to cells will retain the ability toinsert the nucleic acid into a cell, but may be defective for many otherfunctions, such as self-replication.

2. Exemplary Methods

In some aspects, the invention relates to methods for performing MRIusing an intracellular contrast agent that is generated in situ viagenetic instructions and made potent by the sequestering of metal atoms.The sequestered metal atoms are preferably endogenous metal atoms suchas, for example, iron atoms. In certain embodiments, methods of theinvention comprise contacting subject material with a nucleic acidencoding instructions for the synthesis of an intracellular contrastagent, such as a metal binding protein. In such an embodiment, uponinternalization by an appropriate cell, the nucleic acid directsproduction of the metal binding protein which becomes potent as acontrast agent by binding to available metal atoms. In anotherembodiment, the methods of the invention comprise contacting subjectmaterial with a protein or nucleic acid that indirectly affectscontrast, for example, by increasing the amount of metal in the cell orby affecting the expression and/or activity of a metal binding protein.Intracellular contrast agents described herein may be employed in theimaging of essentially any biological material that is capable ofproducing such an agent, including but not limited to: cultured cells,tissues, and living organisms ranging from unicellular organisms tomulticellular organisms (e.g. humans, non-human mammals, othervertebrates, higher plants, insects, nematodes, fungi etc.). While mostbiological systems contain a variety of metals that have potent contrasteffects, it is understood that iron is generally the only such metalthat is sufficiently concentrated to be useful in rendering anintracellular contrast agent potent. However, if desired, material to beimaged may be supplemented with exogenous metal atoms, and suchprotocols will preferably be optimized to reduce deleterious effectscaused by the exogenous metal atoms.

In certain embodiments, the novel contrast technology described hereinmay be employed to investigate the regulation of gene expression insitu. For example, a nucleic acid encoding a contrast protein may beintroduced into a cell, tissue, and/or subject of interest. Those cellshaving appropriate intracellular conditions for expression of thecontrast protein may be distinguished by MRI from cells that do notproduce the contrast protein. In certain embodiments, the nucleic acidencoding the contrast protein is operably linked to a constitutivelyactive regulatory sequence. In further embodiments, the contrast proteinis operably linked to a regulatory sequence so that production of thecontrast protein may be regulated by application of one or moreexogenously controlled conditions, such as temperature changes,concentration of an inducer or repressor, etc. In yet anotherembodiment, the activity of the regulatory sequence is at leastpartially unknown. In a further embodiment, the nucleic acid encoding acontrast protein is not operably linked to a regulatory sequence (or isoperably linked to a weak promoter). This type of “promoterless”construct may be used to identify endogenous sequences that supplyregulatory activity in a manner analogous to an “enhancer trap”.

In certain exemplary embodiments, methods and compositions of theinvention are used to monitor the expression of a transgene of interest,such as a therapeutic transgene. Subject material is contacted with botha transgene of interest, such as a therapeutic transgene, and a nucleicacid construct comprising the coding sequence for a contrast proteinthat is operably linked to a regulatory sequence. In one variation,production of the transgene of interest and production of the contrastprotein are both modulated by functionally similar (optionallyidentical) regulatory sequences. For example, if subject material hasbeen contacted with a transgene under direction of a strong constitutivepromoter, such as certain viral terminal repeat promoters, thenexpression of the gene encoding the contrast protein should also beunder direction of the same promoter or a promoter designed to have asimilar expression pattern. In some variations, the transgene ofinterest is introduced first, and then at a later time the nucleic acidencoding the contrast protein is introduced. In other variations, thenucleic acid encoding the contrast protein is introduced at the sametime as the transgene of interest, and optionally the contrast nucleicacid and the transgene of interest are located on the same vector. Incertain embodiments, the contrast nucleic acid is expressed as atranscriptional fusion with the transgene of interest. In furtherembodiments, the contrast gene and the transgene of interest (or asecond copy thereof) may be expressed as a fusion protein. The fusionprotein approach may be desirable where it is thought that theeffectiveness of the therapeutic transgene is influenced bypost-transcriptional regulation. Subject material may be imaged by MRI,and cells having the contrast protein may be detected and distinguishedfrom cells that do not have the contrast protein. In preferredembodiments, the level of contrast detected by MRI will correlate with,or be indicative of, the level of expression of the transgene ofinterest.

In further exemplary embodiments, methods and compositions of theinvention may be used to investigate the in situ regulatory activity ofa regulatory sequence of interest. Subject material is contacted with anucleic acid encoding a contrast protein, where the nucleic acid isoperably linked to the regulatory sequence of interest. Onceinternalized within an appropriate cell, the contrast gene is expressedat a level that is regulated by the regulatory sequence of interest. Inpreferred embodiments, the level of contrast detected by MRI will becorrelated with the level of activity of the regulatory sequence ofinterest. The regulatory sequence of interest may be essentially anyregulatory sequence, including but not limited to a promoter, anenhancer, an entire promoter/enhancer region, a mutated or altered formof the preceding, or one or more portions of the preceding.

In further exemplary embodiments, the methods described herein may beused to determine whether a physiologically important regulatorysequence is active in situ. For example, the p53 protein is a widelyrecognized regulator of cell proliferation and apoptosis that exerts itsregulatory influences partly in response to DNA damage. Therefore, aconstruct comprising a p53-responsive regulatory sequence operablylinked to a nucleic acid encoding a contrast protein would permitdetection of cells, in situ, in which the p53 regulatory pathway hasbeen activated. Similarly, methods of the invention may be employed toinvestigate, for example, the status of pro-proliferative signalingpathways (e.g. to identify cancerous or pre-cancerous cells), or toassess the status of inflammatory pathways (e.g. in host and/or donortissues in or near transplanted organs), or to non-invasively imagepromoter activation during the course of development, etc. In view ofthis disclosure, one of skill in the art will be able develop myriadrelated methods.

An analogy may be drawn between the traditional reporter gene assaysroutinely performed by biologists, such as assays employingβ-galactosidase (β-Gal) or green fluorescent protein (GFP), and certainembodiments of the present invention. Accordingly, certain methods ofthe invention may be used as an alternative for other commonly usedcell-screening methods. For example, a method for assessing candidatepharmaceuticals may traditionally involve contacting the candidatepharmaceutical with a cell carrying an informative reporter geneconstruct. Now, the standard reporter gene may be replaced with acontrast gene, and the standard detection system may be replaced with anMRI system. While certain embodiments of the present invention may beused to substitute for traditional reporter gene assays, thesetraditional assays are far more limited in their utility. For example,traditional assays use optically-based readout technologies that areineffective in visualizing gene expression deep within intact tissue,and often require histological processing of the biological materials.By contrast, certain embodiments of the present invention employ an MRIcontrast agent as a reporter gene, allowing signal readout deep withinoptically opaque tissues by MRI and, if desired, readouts may beobtained with little or no disruption of the biological function of thesubject material.

In yet another exemplary embodiment, methods and compositions of theinvention may be used to assess the distribution of a vector that hasbeen administered to subject material. For example, a vector designed totransfect an organism may include a nucleic acid encoding a contrastprotein operably linked to a suitable promoter. Optionally, a promoterwill be selected to provide detectable levels of expression in a widerange of tissue types. For example, a strong constitutive promoter mightbe selected. The transfected biological material is imaged by MRI toidentify cells that have been transfected with the vector. Thisexemplary method may be coupled with numerous different methods ofadministering the vector (e.g. introduction into an anatomical region ororgan of particular interest, introduction into the circulatory system,the lymph system, etc.), and may be used to compare vector distributionand transcription levels obtained with each of these approaches. In thecase of delivery systems that are targeted to a particular tissue, theexemplary methodology may be used to confirm or optimize tissuespecificity. As another illustration, the present methods may beemployed to optimize or develop a gene therapy protocol by allowing aninvestigator to determine the location and optionally the level of geneexpression obtained after administration of a particular gene therapysystem.

Many embodiments of the invention pertain to the generation of anartificially induced intracellular contrast agent. In many of thepreceding embodiments, production of the intracellular contrast agent isachieved by introducing a nucleic acid encoding a direct contrastprotein. Generally, production of the contrast agent may be achieved byalternative methods. For example, in situ production of an intracellularcontrast agent may be stimulated by introducing a nucleic acid encodingan indirect contrast agent. An indirect contrast agent may be, forexample, a protein or nucleic acid that regulates iron homeostasis,regulates expression of an endogenous gene coding for a direct contrastagent, and/or regulates the activity of an endogenous protein that mayact as a direct contrast agent, such as, for example, ferritin. Asanother example, production of the contrast agent may be provoked bycontacting the subject material with a composition that elicitsproduction of the contrast agent. For example, cells may be contactedwith an agent, such as an iron source, that causes cells to produceferritin, which is an effective contrast agent. Accordingly, it isunderstood that the invention encompasses agents that are not directcontrast agents and may be neither nucleic acid nor protein but whichnonetheless are useful for inducing in situ production of anintracellular contrast agent.

In certain aspects, nucleic acids of the invention may be introducedinto biological material by using any of a variety of vectors, whethergeneral or organism/tissue/cell-type specific, and in combination withany of a variety of delivery systems, such as for example, liposomes,viral particles, electroporation, etc. In additional aspects, proteinsof the invention may also be administered directly to cells in a varietyof ways, such as liposome fusion, electroporation, attachment to amoiety that is internalized by cells, etc.

In certain embodiments where a nucleic acid encoding a contrast proteinis introduced into cells, it may be desirable to have that gene activeor present in the cells for only a short period of time, or optionallyfor a regulated period of time. If desired, a transient transfectionsystem may be used, and preferably a vector that permits expression for,on average, fewer than one or two days. Alternatively, or inconjunction, gene expression may be controlled by using an externallyregulated promoter, or as a further example, the contrast gene or aportion thereof may be situated with respect to one or morerecombination sites such that activation of a recombinase causesinactivation (or, if preferred, activation) of the nucleic acid encodingthe contrast protein.

Many embodiments of the invention involve the use of nucleic acidsencoding multiple contrast proteins, such as, for example, nucleic acidsencoding heavy and light chains of a mammalian ferritin, or nucleicacids encoding a ferritin and a transferrin receptor.

In certain embodiments, the intracellular contrast agent will be chosenfor safety in the subject material, and where the subject is a humansubject, the intracellular contrast agent is preferably safe for use inhumans.

3. Contrast Agents

In many aspects, as described above, methods of the invention willemploy one or more contrast proteins that generate MRI contrast in vivo.The contrast protein will impart MRI contrast directly, or indirectly,by causing the cell to produce a secondary protein(s) that imparts MRIcontrast. In the case of the direct effector, the contrast protein willtypically form a complex that creates a change in at least one ofrelaxation times T1, T2, and/or T2*, where the change leads to acontrast effect during MRI. Often direct contrast proteins formmetalloprotein complexes. In the case of indirect effectors, thecontrast agent may be, for example, a protein or nucleic acid thatregulates iron homeostasis, regulates expression of an endogenous genecoding for a direct contrast agent, and/or regulates the activity of anendogenous protein that may act as a direct contrast agent, therebyproducing a contrast effect. In certain embodiments, the methodsdescribed herein may involve both direct and indirect contrast agents.In an exemplary embodiment, the methods and/or compositions describedherein comprises an indirect contrast agent that affects ironhomeostasis and a direct contrast agent, such as a metal bindingprotein.

In aspects of the invention employing a metal-binding polypeptide as adirect contrast agent, the metal-binding protein will preferably bind toone or more metals that provide effective contrasting. A variety ofmetals are effective as elements of a contrasting agent, particularlythose with unpaired electrons in the d or f orbitals, such as, forexample, iron (Fe), cobalt (Co), manganese (Mn), nickel (Ni), gadolinium(Gd), etc. As noted above, iron is of particular interest because it ispresent at relatively high levels in mammals and most other organisms,and therefore, detectable accumulations of iron may be generated withoutthe aid of exogenous iron supplementation. Accordingly, preferredmetal-binding proteins of the invention are iron-binding proteins. Inthose embodiments employing a T2 contrast agent, the geometry of metalbinding is not important, but the contrast will tend to be greater whenlarger amounts of metal are concentrated together. In certain preferredembodiments, the effective metal should be bound into a metal-richaggregate, optionally a crystal-like aggregate, greater than 10picometers in diameter, optionally greater than 100 picometers, greaterthan 1 nanometer, greater than 10 nanometers or greater than 100nanometers in diameter. Alternatively the metal-rich aggregate should bein the range of 1-100 nanometers in diameter within the polypeptidecomplex. In a particularly preferred embodiment, the metal-richaggregate exhibits properties of superparamagnetism. When aniron-binding polypeptide is used, it is preferable if the polypeptideretains the iron in the nontoxic Fe(III) oxidation state. Fe(II) is alsoan effective contrasting agent, but Fe(II) may participate in theiron-catalyzed HaberWeiss reaction that yields potentially damaginghydroxyl radicals.

In a preferred embodiment, a direct contrast protein of the inventionhas the following properties: rapid intracellular protein assembly andmetal loading, the tendency to promote formation of a metal-richaggregate that has a large paramagnetic susceptibility, and the abilityto retain the metal in a relatively non-toxic form (e.g. in the case ofiron, the Fe(III) state).

In certain aspects, metal-binding polypeptides may also change thecontrast properties of a cell by perturbing metal metabolism andstimulating the expression of endogenous metal-binding polypeptides thathave contrast effects. This may also lead to an accumulation ordepletion of a particular metal in the cell. For example, transientexpression of high affinity iron-binding proteins may create a temporarydecrease in the intracellular labile iron pool and stimulate productionof transferrin receptor, thereby increasing the net iron uptake into thecell.

Although the exact binding affinity of a metal-binding protein fordifferent metals is not critical, it is generally expected thatpolypeptides with a sub-nanomolar affinity for one or more effectivemetals may be useful, and optionally the polypeptide will have adissociation constant less than 10⁻¹⁵ M, 10⁻²⁰ M, or less for one ormore effective metals. It is understood that many metal binding proteinswill bind to more than one type of metal. For example, lactoferrin willform complexes with metals such as manganese and zinc. Ferritin-ironcomplexes are generally expected to contain some small (perhapsinfinitesimal) amounts of other metals. In general, iron bindingproteins are likely to bind to metals such as manganese, cobalt, zincand chromium, although in vivo the concentration and abundance of ironis so much higher than these other metals that an iron binding proteinwill be primarily associated with iron.

Several exemplary metal-binding polypeptides of the invention areprovided. This is in no way intended to be an exhaustive list, and, inview of the teachings herein, one of skill in the art will be able toidentify or design other useful metal-binding polypeptides.

In certain exemplary embodiments, one or more ferritins may be used as acontrast protein. Ferritins of the invention include any of the group ofdiiron-carboxylate proteins characterized by the tendency to form adimeric or multimeric structure with bound iron and having ahelix-bundle structure comprising an iron-coordinating Glu residue in afirst helix and a Glu-X-X-His motif in a second. Certain ferritinsmaintain bound iron in a primarily Fe(III) form. A list of exemplaryferritins is provided in Table 1. This list is intended to provideexamples and is not intended to be comprehensive. Many known ferritinsare not included, and it is understood that most vertebrate species willhave a form, of ferritin that can be used as a contrast agent. In viewof this specification, one of ordinary skill in the art will be able toidentify additional ferritin homologs. In certain embodiments, aferritin for use as a contrasting agent should have at least 50%identity with the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO:4,and optionally at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%identity with the amino acid sequence of SEQ ID NO:2 and/or SEQ ID NO:4.

In many embodiments, methodologies of the invention will employ avertebrate ferritin as a contrast agent. Vertebrate ferritins typicallyform a large complex that assembles in a shell to delimit a cavity whereiron is accumulated in a mineral and compact form. Most mammalianferritins are composed of two subunit types, the H- and L-chains.Typically the endogenous mRNAs for the two chains have nearly identicaliron-responsive elements (IREs) close to the 5′ termini that regulateferritin translation by binding to iron-regulatory proteins (IRPs). Whendesigning nucleic acid constructs for the ectopic expression offerritins, it will often be desirable to omit or otherwise disrupt theIRE sequences. Contacting cultured cells with an elevated ironconcentration typically causes a strong up-regulation of both the L- andH-chains, whereas treatment with iron chelating agents, such asdesferrioxamine, suppresses ferritin production. Preferred ferritins ofthe invention catalyze both an iron oxidation step from the Fe(II) formto the Fe(III) form and also catalyze the nucleation and growth of aniron mineral core. In the case of ferritins composed of multiplesubunits, it will typically be desirable to express all subunits at astoichiometry approximating that found in the native complexes. However,it is notable that a wide range of subunit ratios will typically beeffective. For example, human H chain is capable of forming ahomopolymer that binds iron. Excess ferritin resulting fromoverexpression is typically degraded inside the cell, and the primarydecay product is hemosiderin deposits; these are also effective ascontrast agents.

TABLE 1 Exemplary Ferritin Proteins and Nucleic Acids Amino Acid NucleicAcid Sequence Sequence Name (Acc. No.) (Acc. No.) ferritin, heavypolypeptide 1 AAH16009.1 BC016009.1 [Homo sapiens] ferritin, lightpolypeptide XP_050469.1 XM_050469.1 [Homo sapiens] ferritin heavy chain[Mus NP_034369.1 NM_010239.1 musculus] ferritin light chain 1 [MusNP_034370.1 NM_010240.1 musculus] ferritin light chain 2 [MusNP_032075.1 NM_008049.1 musculus] ferritin subunit H [Rattus NP_036980.1NM_012848.1 norvegicus] ferritin light chain 1 [Rattus NP_071945.1NM_022500.1 norvegicus] ferritin heavy chain [Cavia BAB70615.1AB073371.1 porcellus] ferritin light chain [Cavia AAF36408.1 AF233445_1porcellus] ferritin heavy chain [rabbit] P25915 ferritin light chain[rabbit] S01239 ferritin H subunit [Bos BAA24818.1 AB003093.1 taurus]ferritin L subunit [Bos BAA24819.1 AB003094.1 taurus] ferritin heavychain [Gallus A26886 gallus] ferritin [Canis familiaris] AAK82992.1AF285177.1 ferritin H chain [Macaca AAF98711.1 AF162481_1 mulatta]ferritin heavy chain FRXL [Xenopus laevis] ferritin heavy chain [DanioAAG37837.1 AF295373_1 rerio] yolk ferritin [Paragonimus AAG17056.1AF188720_1 westermani] ferritin [Taenia saginata] CAA65097.1 26 kDaferritin subunit AAG41120.1 AF142340.1 [Galleria mellonella] nonhemeiron-containing NP_207447.1 NC_000915.1 ferritin (pfr) [Helicobacterpylori 26695] ferritin [Glycine max] AAL09920.1 AY049920.1

In a further embodiment, a metal binding protein of the invention is ametal scavenger, defined as a protein that binds metal with very highaffinity through a siderophore. Such proteins may be used as contrastagents. While not wishing to be bound to a mechanism, it is expectedthat such proteins will act primarily as indirect contrast agents. Forexample, iron scavenging proteins expressed in a cell may scavenge andtightly bind iron from the labile iron pool within the intracellularspace. Thus MRI contrast may be enhanced by a combination of theiron-bound chelate itself and the additional iron that is sequesteredand stored as a result of the cell's own iron regulation mechanisms.Exemplary siderophores that may be present in metal scavenging proteinsinclude hemoglobin, and any other agent that provides an octahederalcoordination sphere for the iron, usually formed-by six oxygen atoms. Ingeneral these fall into two categories: (a) catechols such asenterobactin which comprises a cyclic structure composed of threemolecules of 2,3-dihydroxy-N-benzoyl serine. Further examples includeagents wherein the serine is substituted with either a glycine or athreonine. Also included herein are catechol siderophores having linearrather than cyclic structures such as pseudobactin; (b) Hydroxamatescomprise a large and variable group having either cyclic or linearpeptides containing various types of hydroxamic acids. Common examplesinclude ferrichrome, ferrioxamine, and aerobactin. Further examplesinclude plant siderophores such as phytosiderophore. Exemplary metalscavenging proteins include ferric binding proteins of the siderophilinfamily, such as mammalian transferring, ovotransferin, lactoferrins,melanotransferrin, sertoli transferrin, neurotransferrin, mucosaltransferrin, and bacterial transferring, such as those found inHaemophilus influenzae, Neisseria gonorrhoeae, and Neisseriameningitidis.

In further embodiments, an iron regulatory protein (IRP) may be used asa contrast protein. IRPs are iron-regulating RNA binding proteins thatmodulate synthesis of proteins that function in the uptake (e.g.transferrin receptor), utilization (e.g. erythroid 5-aminolevulinatesynthase) or storage (e.g. H-ferritin and L-ferritin) of iron. Proteinsregulated by IRPs are encoded by mRNAs that include one or morestem-loop motifs, termed an Iron Responsive Element (IRE). Under lowiron conditions, IRPs bind to IREs and modulate the stability ortranslation of the affected mRNA. In general, when an IRE is positionedin the 5′ UTR region of an mRNA (e.g. the ferritins), the IRP blockstranslation, causing decreased protein production in low ironconditions. When an IRE is positioned in the 3′ UTR (e.g. transferrinreceptor), the IRP typically stabilizes the mRNA, thereby increasingproduction of the gene product in response to low iron conditions. Micehaving a targeted deletion of the gene encoding IRP2 show significantaccumulations of iron in neural tissues (LaVaute et al., 2001, Nat.Genet. 27(2):209-14). Accordingly, manipulation of IRPs by, for example,antisense or RNAi methodologies may provide contrast effects. IRPs ofthe invention will typically have the ability to bind to IREs in aniron-regulated manner. Preferred IRPs of the invention will bevertebrate IRPs such as: human IRP1 (Ace. Nos. P21399 and Z11559), humanIRP2 (Ace. Nos. AAA69901 and M58511), rat IRE-BP1 (Ace. Nos. Q63270 andL23874), mouse IRE-BP1 (Ace. Nos. P28271 and X61147), chicken IRE-BP(Ace. No. Q90875 and D16150), etc. In general, it will be desirable toemploy an IRP that binds to the IREs of the subject biological material,and in certain embodiments, this may be accomplished by using an IRPthat is derived from the subject species. In certain aspects of theinvention, a contrast protein comprises an amino acid sequence at least60% identical to that of human IRP1 and/or IRP2, and optionally at least70%, 80%, 90%, 95%, 98%, 99% or 100% identical.

In further aspects, a contrast protein of the invention may be one thatperturbs cellular iron homeostasis. For example, a transferrin receptorprotein, and/or a molecule that regulates the expression and/or functionof a transferrin receptor protein, may be used as a contrast agent.Transferrin receptor mediates the receptor mediated endocytosis of theiron-carrying protein transferrin and thereby mediates cellular ironuptake. Therefore, in one embodiment of the invention, the level and/oractivity of a transferrin receptor in targeted cells may be modulated soas to produce an increase in cellular iron uptake thereby causing thecell to produce ferritin. The end result will be an accumulation ofexcess ferritin that will yield MRI contrast. Exemplary transferrinreceptors include SEQ ID Nos: 16, 18, 20 and 22. In certain aspects ofthe invention, a contrast protein comprises an amino acid sequence atleast 60% identical to that of human transferrin receptor 1 and/or humantransferrin receptor 2, and optionally at least 70%, 80%, 90%, 95%, 98%,99% or 100% identical, and preferably retains transferrin receptoractivity.

In further embodiments, contrast proteins of the invention may beengineered, by for example, employing techniques of molecular biology.For example, it is possible to modify the structure of the subjectcontrast proteins for such purposes as enhancing contrast efficacy,stability (e.g., increased or decreased resistance to proteolyticdegradation in vivo), antigenicity, or safety, among othercharacteristics. Such modified proteins can be produced, for instance,by amino acid substitution, deletion, or addition. In addition, simplevariants of any of the proteins discussed herein may be obtained byconservative substitution. For instance, it is reasonable to expect thatan isolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(i.e. conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids are can bedivided into four families: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer,W. H. Freeman and Co., 1981).

This invention further contemplates methods of generating sets ofcombinatorial mutants of the subject contrast proteins, as well asfunctional truncation mutants. The purpose of screening suchcombinatorial libraries is to generate, for example, engineered contrastproteins with any number of desirable qualities such as those mentionedabove.

There are many ways by which the library of potential engineeredcontrast proteins can be generated. Chemical synthesis of a degenerategene sequence can be carried out in an automatic DNA synthesizer, andthe synthetic genes then be ligated into an appropriate gene forexpression. The purpose of a degenerate set of genes is to provide, inone mixture, all of the sequences encoding the desired set of potentialcontrast protein sequences. Such techniques have been employed in thedirected evolution of other proteins (see, for example, Scott et al.,(1990) Science 249:386-390; Roberts et al., (1992) PNAS USA89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,(1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, engineered contrast proteins can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (see e.g. Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993), by linker scanning mutagenesis(Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol.Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); bysaturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCRmutagenesis (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or byrandom mutagenesis, including chemical mutagenesis, etc. (Miller et al.,(1992) A Short Course in Bacterial Genetics, CSHL Press, Cold SpringHarbor, N.Y.; and Greener et al., (1994) Strategies in Mol Biol7:32-34).

Whether a change in the amino acid sequence of a polypeptide results ina functional homologue can be readily determined by assessing theability of the variant polypeptide to, for example, bind the desiredmetal, produce sufficient MRI contrast in cells, and produce reducedcell toxicity.

In further aspects, any combination of contrast proteins may employed toobtain the desired contrast effects.

4. Constructs and Vectors

In certain aspects, the invention provides vectors and nucleic acidconstructs comprising nucleic acids encoding one or more contrastagents. Other features of the vector or construct will generally bedesigned to supply desirable characteristics depending on how thecontrast agent is to be generated and used. Exemplary desirablecharacteristics include but are not limited to, gene expression at adesired level, gene expression that is reflective of the expression of adifferent gene, easy clonability, transient or stable gene expression insubject cells, etc.

In certain aspects, it is desirable to use a vector that providestransient expression of the contrast agent. Such vectors will generallybe unstable inside a cell, such that the nucleic acids necessary forexpression of the contrast agent are lost after a relatively shortperiod of time. Optionally, transient expression may be effected bystable repression. Exemplary transient expression vectors may bedesigned to provide gene expression for an average time of hours, days,weeks, or perhaps months. Often transient expression vectors do notrecombine to integrate with the stable genome of the host. Exemplarytransient expression vectors include: adenovirus-derived vectors,adeno-associated viruses, herpes simplex derived vectors, hybridadeno-associated/herpes simplex viral vectors, influenza viral vectors,especially those based on the influenza A virus, and alphaviruses, forexample the Sinbis and semliki forest viruses.

In some aspects the invention provides a vector or construct comprisinga readily clonable nucleic acid encoding a contrast protein. Forexample, the coding sequence may be flanked by a polylinker on one orboth sides. Polylinkers are useful for allowing one of skill in the artto readily insert the coding sequence in a variety of different vectorsand constructs as required. In another example, the coding sequence maybe flanked by one or more recombination sites. A variety of commerciallyavailable cloning systems use recombination sites to facilitate movementof the desired nucleic acid into different vectors. For example, theInvitrogen Gateway™ technology utilizes a phage lambda recombinaseenzyme to recombine target nucleic acids with a second nucleic acid.Each nucleic acid is flanked with appropriate lambda recognitionsequence, such as attL or attB. In other variations, a recombinase suchas topoisomerase I may be used with nucleic acids flanked by theappropriate recognition sites. For example, the Vaccinia virustopoisomerase I protein recognizes a (C/T)CCTT sequence. Theserecombination systems permit rapid shuffling of flanked cassettes fromone vector to another as needed. A construct or vector may include bothflanking polylinkers and flanking recombination sites, as desired.

In certain aspects, the contrast gene is operably linked to a promoter.The promoter may for example, be a strong or constitutive promoter, suchas the early and late promoters of SV40, or adenovirus orcytomegalovirus immediate early promoter. Optionally it may be desirableto use an externally regulated promoter, such as a tet promoter,IPTG-regulated promoters (GAL4, Plac), or the trp system. In view ofthis specification, one of skill in the art will readily identify otheruseful promoters depending on the downstream use. For example, theinvention may utilize exemplary promoters such as the T7 promoter whoseexpression is directed by T7 RNA polymerase, the major operator andpromoter regions of phage lambda, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast a-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. In addition, as noted above, it may be desirableto have a contrast gene operably linked to a promoter that providesuseful information about the condition of the cell in which it issituated. In certain embodiments, it is anticipated that it will bedesirable to achieve a concentration of contrast protein within targetcells that permits detection above background noise, and with certaindetection systems this will translate into a protein concentration of atleast 1 nM or at least 10 nM.

Vectors of the invention may be essentially any nucleic acid designed tointroduce and/or maintain a contrast gene in a cell or virus. ThepcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) may be used. Othervector systems suitable for gene therapy are described below.

5. Cells, Organized Cell Cultures, and Tissues

In many aspects, the invention provides cells, organized cell cultures,and tissues comprising a nucleic acid that encodes a contrast agent.Methods for generating transformed or transfected cells are widely knownin the art, and it is anticipated that methods described herein may beused with essentially any cell type of interest, including but notlimited to bacterial, fungal, plant and animal cells. Preferredembodiments of the invention employ mammalian cells. Cells of particularinterest may include transformed cells or other cells that either arepart of a tumor or are useful as a model for cancer in vitro, stem orprogenitor cells, and cells prepared for a cell therapy for a patient.Cells of the invention may be cultured cells, cell lines, cells situatedin tissues and/or cells that are part of an organism.

It is further anticipated that cells may be used to generate organizedcell cultures (i.e. cell cultures developing a non-random structure) andto generate organs or organ-like structures for transplant intosubjects. It may be useful to non-invasively monitor some aspect of geneexpression in such cells, or to otherwise provide MRI contrast in suchcells. For example, muscle progenitor cells may be used to developmuscle-like organs for administration to injured muscle or foradministration as a packet of cells that produce a therapeutic protein(see e.g. U.S. Pat. Nos. 5,399,346; 6,207,451; 5,538,722). Other cellculture methods have been used to produce neural, pancreatic, liver andmany other organ types for transplant (see e.g. U.S. Pat. Nos.6,146,889; 6,001,647; 5,888,705; 5,851,832 and PCT publication nos. WO00/36091; WO 01/53461; WO 01/21767). Cells of this nature may be stablytransfected with a contrast gene at an early stage of culture, or theorganized culture may be transiently or stably transfected at a laterpoint in culture to assess some aspect of cell function. Transfectedcells may be administered to subjects in order to deliver a geneproduct, and this methodology is effective as an ex vivo gene therapy orcell therapy method. A nucleic acid encoding a contrast protein may beintroduced into such cells and administered to a subject in order tomonitor gene expression or viability of the administered cells. Cellstransfected with the gene adenosine deaminase have been delivered topatients as an ex vivo gene therapy cure for Severe CombinedImmunodeficiency Syndrome (SCID) (Cavazzana-Calvo et al., 2000, Science288(5466):669-72).

6. Nucleic Acids for Delivery to Organisms and In vitro Tissues

Instead of ex vivo modification of cells, in many situations one maywish to modify cells in vivo. For this purpose, various techniques havebeen developed for modification of target tissue and cells in vivo. Anumber of viral vectors have been developed, such as described above,which allow for transfection and, in some cases, integration of thevirus into the host. See, for example, Dubensky et al. (1984) Proc.Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science243,375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86,3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 andFerry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Thevector may be administered by injection, e.g. intravascularly orintramuscularly, inhalation, or other parenteral mode. Non-viraldelivery methods such as administration of the DNA via complexes withliposomes or by injection, catheter or biolistics may also be used.Generally, in human subjects, it will be preferable to design thenucleic acid and/or the delivery system to provide transient expressionof the nucleic acid encoding the contrast agent.

In general, the manner of introducing the nucleic acid will depend onthe nature of the tissue, the efficiency of cellular modificationrequired, the number of opportunities to modify the particular cells,the accessibility of the tissue to the nucleic acid composition to beintroduced, and the like. The DNA introduction need not result inintegration. In fact, non-integration often results in transientexpression of the introduced DNA, and transient expression is oftensufficient or even preferred.

Any means for the introduction of polynucleotides into mammals, human ornon-human, may be adapted to the practice of this invention for thedelivery of the various constructs of the invention into the intendedrecipient. In one embodiment of the invention, the nucleic acidconstructs are delivered to cells by transfection, i.e., by delivery of“naked” nucleic acid or in a complex with a colloidal dispersion system.A colloidal system includes macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. An exemplarycolloidal system of this invention is a lipid-complexed orliposome-formulated DNA. In the former approach, prior to formulation ofDNA, e.g., with lipid, a plasmid containing a transgene bearing thedesired DNA constructs may first be experimentally optimized forexpression (e.g., inclusion of an intron in the 5′ untranslated regionand elimination of unnecessary sequences (Felgner, et al., Ann NY AcadSci 126-139, 1995). Formulation of DNA, e.g. with various lipid orliposome materials, may then be effected using known methods andmaterials and delivered to the recipient mammal. See, e.g., Canonico etal, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol268; Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No.5,679,647 by Carson et al.

Optionally, liposomes or other colloidal dispersion systems aretargeted. Targeting can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs, which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. A certain level of targeting maybe achieved through the mode of administration selected.

In certain variants of the invention, the nucleic acid constructs aredelivered to cells, and particularly cells in an organism or a culturedtissue, using viral vectors. The transgene may be incorporated into anyof a variety of viral vectors useful in gene therapy, such asrecombinant retroviruses, adenovirus, adeno-associated virus (AAV),herpes simplex derived vectors, hybrid adeno-associated/herpes simplexviral vectors, influenza viral vectors, especially those based on theinfluenza A virus, and alphaviruses, for example the Sinbis and semlikiforest viruses, or recombinant bacterial or eukaryotic plasmids. Thefollowing additional guidance on the choice and use of viral vectors maybe helpful to the practitioner. As described in greater detail below,such embodiments of the subject expression constructs are specificallycontemplated for use in various in vivo and ex vivo gene therapyprotocols.

A. Herpes Virus Systems

A variety of herpes virus-based vectors have been developed forintroduction of genes into mammals. For example, herpes simplex virustype 1 (HSV-1) is a human neurotropic virus of particular interest forthe transfer of genes to the nervous system. After infection of targetcells, herpes viruses often follow either a lytic life cycle or a latentlife cycle, persisting as an intranuclear episome. In most cases,latently infected cells are not rejected by the immune system. Forexample, neurons latently infected with HSV-1 function normally and arenot rejected. Some herpes viruses possess cell-type specific promotersthat are expressed even when the virus is in a latent form.

A typical herpes virus genome is a linear double stranded DNA moleculeranging from 100 to 250 kb. HSV-1 has a 152 kb genome. The genome mayinclude long and short regions (termed UL and US, respectively) whichare linked in either orientation by internal repeat sequences (IRL andIRS). At the non-linker end of the unique regions are terminal repeats(TRL and TRS). In HSV-1, roughly half of the 80-90 genes arenon-essential, and deletion of non-essential genes creates space forroughly 40-50 kb of foreign DNA (Glorioso et al, 1995). Two latencyactive promoters which drive expression of latency activated transcriptshave been identified and may prove useful for vector transgeneexpression (Marconi et al, 1996).

HSV-1 vectors are available in amplicons and recombinant HSV-1 virusforms. Amplicons are bacterially produced plasmids containing OriC, anEscherichia coli origin of replication, OriS (the HSV-1 origin ofreplication), HSV-1 packaging sequence, the transgene under control ofan immediate-early promoter & a selectable marker (Federoff et al,1992). The amplicon is transfected into a cell line containing a helpervirus (a temperature sensitive mutant) which provides all the missingstructural and regulatory genes in trans. More recent amplicons includean Epstein-Barr virus derived sequence for plasmid episomal maintenance(Wang & Vos, 1996). Recombinant viruses are made replication deficientby deletion of one the immediate-early genes e.g. ICP4, which isprovided in trans. Deletion of a number of immediate-early genessubstantially reduces cytotoxicity and allows expression from promotersthat would be silenced in the wild type latent virus. These promotersmay be of use in directing long term gene expression.Replication-conditional mutants replicate in permissive cell lines.Permissive cell lines supply a cellular enzyme to complement for a viraldeficiency. Mutants include thymidine kinase (During et al, 1994),ribonuclease reductase (Kramm et al, 1997), UTPase, or theneurovirulence factor g34.5 (Kesari et al, 1995). These mutants areparticularly useful for the treatment of cancers, killing the neoplasticcells which proliferate faster than other cell types (Andreansky et al,1996, 1997). A replication-restricted HSV-1 vector has been used totreat human malignant mesothelioma (Kucharizuk et al, 1997). In additionto neurons, wild type HSV-1 can infect other non-neuronal cell types,such as skin (Al-Saadi et al, 1983), and HSV-derived vectors may beuseful for delivering transgenes to a wide array of cell types. Otherexamples of herpes virus vectors are known in the art (U.S. Pat. No.5,631,236 and WO 00/08191).

B. Adenoviral Vectors

A viral gene delivery system useful in the present invention utilizesadenovirus-derived vectors. Knowledge of the genetic organization ofadenovirus, a 36 kB, linear and double-stranded DNA virus, allowssubstitution of a large piece of adenoviral DNA with foreign sequencesup to 8 kB. In contrast to retrovirus, the infection of adenoviral DNAinto host cells does not result in chromosomal integration becauseadenoviral DNA can replicate in an episomal manner without potentialgenotoxicity. Also, adenoviruses are structurally stable, and no genomerearrangement has been detected after extensive amplification.Adenovirus can infect virtually all epithelial cells regardless of theircell cycle stage. In addition, adenoviral vector-mediated transfectionof cells is often a transient event. A combination of immune responseand promoter silencing appears to limit the time over which a transgeneintroduced on an adenovirus vector is expressed.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range, and high infectivity. The virus particle isrelatively stable and amenable to purification and concentration, and asabove, can be modified so as to affect the spectrum of infectivity.Additionally, adenovirus is easy to grow and manipulate and exhibitsbroad host range in vitro and in vivo. This group of viruses can beobtained in high titers, e.g., 10⁹-10¹¹ plaque-forming unit (PFU)/ml,and they are highly infective. Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al., supra; Haj-Ahmand andGraham (1986) J. Virol. 57:267). Most replication-defective adenoviralvectors currently in use and therefore favored by the present inventionare deleted for all or parts of the viral E1 and E3 genes but retain asmuch as 80% of the adenoviral genetic material (see, e.g., Jones et al.,(1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methodsin Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991)vol. 7. pp. 109-127). Expression of the inserted polynucleotide of theinvention can be under control of, for example, the E1A promoter, themajor late promoter (MLP) and associated leader sequences, the viral E3promoter, or exogenously added promoter sequences.

The genome of an adenovirus can be manipulated such that it encodes agene product of interest, but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle (see, for example, Berkneret al., (1988) BioTechniques 6:616; Rosenfeld et al., (1991) Science252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155). Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 dl324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known tothose skilled in the art.

Adenoviruses can be cell type specific, i.e., infect only restrictedtypes of cells and/or express a transgene only in restricted types ofcells. For example, the viruses may be engineered to comprise a geneunder the transcriptional control of a transcription initiation regionspecifically regulated by target host cells, as described e.g., in U.S.Pat. No. 5,698,443, by Henderson and Schuur, issued Dec. 16, 1997. Thus,replication competent adenoviruses can be restricted to certain cellsby, e.g., inserting a cell specific response element to regulate asynthesis of a protein necessary for replication, e.g., E1A or E1B.

DNA sequences of a number of adenovirus types are available fromGenbank. For example, human adenovirus type 5 has GenBank AccessionNo.M73260. The adenovirus DNA sequences may be obtained from any of the42 human adenovirus types currently identified. Various adenovirusstrains are available from the American Type Culture Collection,Rockville, Md., or by request from a number of commercial and academicsources. A transgene as described herein may be incorporated into anyadenoviral vector and delivery protocol, by restriction digest, linkerligation or filling in of ends, and ligation.

Adenovirus producer cell lines can include one or more of the adenoviralgenes E1, E2a, and E4 DNA sequence, for packaging adenovirus vectors inwhich one or more of these genes have been mutated or deleted aredescribed, e.g., in PCT/US95/15947 (WO 96/18418) by Kadan et al.;PCT/US95/07341 (WO 95/346671) by Kovesdi et al.; PCT/FR94/00624(WO94/28152) by Imler et al.;PCT/FR94/00851 (WO 95/02697) by Perrocaudetet al., PCT/US95/14793 (WO96/14061) by Wang et al.

C. AAV Vectors

Yet another viral vector system useful for delivery of the subjectpolynucleotides is the adeno-associated virus (AAV). Adeno-associatedvirus is a naturally occurring defective virus that requires anothervirus, such as an adenovirus or a herpes virus, as a helper virus forefficient replication and a productive life cycle. (For a review, seeMuzyczka et al., Curr. Topics in Micro. and Immunol. (1992) 158:97-129).

AAV has not been associated with the cause of any disease. AAV is not atransforming or oncogenic virus. AAV integration into chromosomes ofhuman cell lines does not cause any significant alteration in the growthproperties or morphological characteristics of the cells. Theseproperties of AAV also recommend it as a potentially useful human genetherapy vector.

AAV is also one of the few viruses that may integrate its DNA intonon-dividing cells, e.g., pulmonary epithelial cells, and exhibits,ahigh frequency of stable integration (see for example Flotte et al.,(1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al.,(1989) J. Virol. 63:3822-3828; and McLaughlin et al., (1989) J. Virol.62:1963-1973). Vectors containing as little as 300 base pairs of AAV canbe packaged and can integrate. Space for exogenous DNA is limited toabout 4.5 kb. An AAV vector such as that described in Tratschin et al.,(1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA intocells. A variety of nucleic acids have been introduced into differentcell types using AAV vectors (see for example Hermonat et al., (1984)PNAS USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol.4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39;Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993)J. Biol. Chem. 268:3781-3790).

The AAV-based expression vector to be used typically includes the 145nucleotide AAV inverted terminal repeats (ITRs) flanking a restrictionsite that can be used for subcloning of the transgene, either directlyusing the restriction site available, or by excision of the transgenewith restriction enzymes followed by blunting of the ends, ligation ofappropriate DNA linkers, restriction digestion, and ligation into thesite between the ITRs. The capacity of AAV vectors is usually about 4.4kb (Kotin, R. M., Human Gene Therapy 5:793-801, 1994 and Flotte, et al.J. Biol. Chem. 268:3781-3790, 1993).

AAV stocks can be produced as described in Hermonat and Muzyczka (1984)PNAS 81:6466, modified by using the pAAV/Ad described by Samulski et al.(1989) J. Virol. 63:3822. Concentration and purification of the viruscan be achieved by reported methods such as banding in cesium chloridegradients, as was used for the initial report of AAV vector expressionin vivo (Flotte, et al. J. Biol. Chem. 268:3781-3790, 1993) orchromatographic purification, as described in O'Riordan et al.,WO97/08298. Methods for in vitro packaging AAV vectors are alsoavailable and have the advantage that there is no size limitation of theDNA packaged into the particles (see, U.S. Pat. No. 5,688,676, by Zhouet al., issued Nov. 18, 1997). This procedure involves the preparationof cell free packaging extracts.

D. Hybrid Adenovirus-AAV Vectors

Hybrid Adenovirus-AAV vectors have been generated and are typicallyrepresented by an adenovirus capsid containing a nucleic acid comprisinga portion of an adenovirus, and 5′ and 3′ inverted terminal repeatsequences from an AAV which flank a selected transgene under the controlof a promoter. See e.g. Wilson et al, International Patent ApplicationPublication No. WO 96/13598. This hybrid vector is characterized by hightiter transgene delivery to a host cell and the ability to stablyintegrate the transgene into the host cell chromosome in the presence ofthe rep gene. This virus is capable of infecting virtually all celltypes (conferred by its adenovirus sequences) and stable long termtransgene integration into the host cell genome (conferred by its AAVsequences).

The adenovirus nucleic acid sequences employed in this vector can rangefrom a minimum sequence amount, which requires the use of a helper virusto produce the hybrid virus particle, to only selected deletions ofadenovirus genes, which deleted gene products can be supplied in thehybrid viral process by a packaging cell. For example, a hybrid viruscan comprise the 5′ and 3′ inverted terminal repeat (ITR) sequences ofan adenovirus (which function as origins of replication). The leftterminal sequence (5′) sequence of the Ad5 genome that can be used spansbp 1 to about 360 of the conventional adenovirus genome (also referredto as map units 0-1) and includes the 5′ ITR and the packaging/enhancerdomain. The 3′ adenovirus sequences of the hybrid virus include theright terminal 3′ ITR sequence which is about 580 nucleotides (about bp35,353-end of the adenovirus, referred to as about map units 98.4-100).

For additional detailed guidance on adenovirus and hybrid adenovirus-AAVtechnology which may be useful in the practice of the subject invention,including methods and materials for the incorporation of a transgene,the propagation and purification of recombinant virus containing thetransgene, and its use in transfecting cells and mammals, see alsoWilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, and referencescited therein.

E. Retroviruses

In order to construct a retroviral vector, a nucleic acid of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and psi components is constructed (Mann et al.(1983) Cell 33:153). When a recombinant plasmid containing a human cDNA,together with the retroviral LTR and psi sequences is introduced intothis cell line (by calcium phosphate precipitation for example), the psisequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein (1988) “Retroviral Vectors”, In: Rodriguezand Denhardt ed. Vectors: A Survey of Molecular Cloning Vectors andtheir Uses. Stoneham:Butterworth; Temin, (1986) “Retrovirus Vectors forGene Transfer: Efficient Integration into and Expression of ExogenousDNA in Vertebrate Cell Genome”, In: Kucherlapati ed. Gene Transfer. NewYork: Plenum Press; Mann et al., 1983, supra). The media containing therecombinant retroviruses is then collected, optionally concentrated, andused for gene transfer. Retroviral vectors are able to infect a broadvariety of cell types. Integration and stable expression require thedivision of host cells (Paskind et al. (1975) Virology 67:242). Thisaspect is particularly relevant for the treatment of PVR, since thesevectors allow selective targeting of cells which proliferate, i.e.,selective targeting of the cells in the epiretinal membrane, since theseare the only ones proliferating in eyes of PVR subjects.

A major prerequisite for the use of retroviruses is to ensure the safetyof their use, particularly with regard to the possibility of the spreadof wild-type virus in the cell population. The development ofspecialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A.D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding aprotein of the present invention, e.g., a transcriptional activator,rendering the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.A preferred retroviral vector is a pSR MSVtkNeo (Muller et al. (1991)Mol. Cell Biol. 11:1785 and pSR MSV(XbaI) (Sawyers et al. (1995) J. Exp.Med. 181:307) and derivatives thereof. For example, the unique BamHIsites in both of these vectors can be removed by digesting the vectorswith BamHI, filling in with Klenow and religating to produce pSMTN2 andpSMTX2, respectively, as described in PCT/US96/09948 by Clackson et al.Examples of suitable packaging virus lines for preparing both ecotropicand amphotropic retroviral systems include Crip, Cre, 2 and Am.

Retroviruses, including lentiviruses, have been used to introduce avariety of genes into many different cell types, including neural cells,epithelial cells, retinal cells, endothelial cells, lymphocytes,myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (seefor example, review by Federico (1999) Curr. Opin. Biotechnol. 10:448;Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988)PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018;Armentano et al., (1990) PNAS USA 87:6141-6145; Huber et al., (1991)PNAS USA 88:8039-8043; Ferry et al., (1991) PNAS USA 88:8377-8381;Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al.,(1992) PNAS USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy3:641-647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwu et al.,(1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan et al., (1992)J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology163:251-254); or coupling cell surface ligands to the viral env proteins(Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be inthe form of the chemical cross-linking with a protein or other variety(e.g. lactose to convert the env protein to an asialoglycoprotein), aswell as by generating fusion proteins (e.g. single-chain antibody/envfusion proteins). This technique, while useful to limit or otherwisedirect the infection to certain tissue types, and can also be used toconvert an ecotropic vector in to an amphotropic vector.

F. Other Viral Systems

Other viral vector systems that can be used to deliver a polynucleotideof the invention have been derived from vaccinia virus, alphavirus,poxvirus, arena virus, polio virus, and the like. Such vectors offerseveral attractive features for various mammalian cells. (Ridgeway(1988) In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey ofmolecular cloning vectors and their uses. Stoneham: Butterworth;Baichwal and Sugden (1986) In: Kucherlapati R, ed. Gene transfer. NewYork: Plenum Press; Coupar et al. (1988) Gene, 68:1-10; Walther andStein (2000) Drugs 60:249-71; Timiryasova et al. (2001) J Gene Med3:468-77; Schlesinger (2001) Expert Opin Biol Ther 1:177-91; Khromykh(2000) Curr Opin Mol Ther 2:555-69; Friedmann (1989) Science,244:1275-1281; Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra;Coupar et al., 1988; Horwich et al. (1990) J. Virol., 64:642-650).

7. Transgenic Animals

While the techniques described herein may be used to deliver nucleicacids to human or animal subjects, other methods are available togenerate non-human transgenic animals incorporating a recombinantnucleic acid encoding a contrast protein.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, ME). Preferred strains such as C57BL/6or DBA/1 may be selected. The line(s) used to practice this inventionmay themselves be transgenics, and/or may be knockouts (i.e., obtainedfrom animals which have one or more genes partially or completelysuppressed).

In one embodiment, the construct comprising a nucleic acid encoding acontrast protein is introduced into a single stage embryo. The zygote isthe best target for microinjection. In the mouse, the male pronucleusreaches the size of approximately 20 micrometers in diameter whichallows reproducible injection of 1-2 pl of DNA solution. The use ofzygotes as a target for gene transfer has a major advantage in that inmost cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear. At about this time, the nucleotide sequence comprisingthe transgene is introduced into the female or male pronucleus asdescribed below. In some species such as mice, the male pronucleus ispreferred. It is most preferred that the exogenous genetic material beadded to the male DNA complement of the zygote prior to its beingprocessed by the ovum nucleus or the zygote female pronucleus. It isthought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

Thus, it is preferred that the exogenous genetic material be added tothe male complement of DNA or any other complement of DNA prior to itsbeing affected by the female pronucleus. For example, the exogenousgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcan then be added to the ovum or the decondensed sperm could be added tothe ovum with the transgene constructs being added as soon as possiblethereafter.

Introduction of the transgene nucleotide sequence into the embryo may beaccomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, i.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the euploid number of the organismfrom which either gamete originated.

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA sequenceswill vary depending upon the particular zygote and functions of theexogenous genetic material and will be readily apparent to one skilledin the art, because the genetic material, including the exogenousgenetic material, of the resulting zygote must be biologically capableof initiating and maintaining the differentiation and development of thezygote into a functional organism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional. Asregards the present invention, there will often be an advantage tohaving more than one functioning copy of each of the inserted exogenousDNA sequences to enhance the phenotypic expression of the exogenous DNAsequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for thepresence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents. Alternatively, MRI can be used tovisualize transgene expression.

An alternative method for generating transgenic animals involves the invivo or ex vivo (in vitro) transfection of male animal germ cells with adesired nucleic acid (see e.g., U.S. Pat. No. 6,316,692). In oneapproach, the nucleic acid is delivered in situ to the gonad of theanimal (in vivo transfection). The transfected germ cells are allowed todifferentiate in their own milieu, and then animals exhibitingintegration of the nucleic acid into the germ cells are selected. Theselected animals may be mated, or their sperm utilized for inseminationor in vitro fertilization to produce transgenic progeny. The selectionmay take place after biopsy of one or both gonads, or after examinationof the animal's ejaculate to confirm the incorporation of the desirednucleic acid sequence. Alternatively, male germ cells may be isolatedfrom a donor animal and transfected, or genetically altered in vitro.Following this genetic manipulation, transfected germ cells are selectedand transferred to the testis of a suitable recipient animal. Beforetransfer of the germ cells, the recipient testis are generally treatedin one, or a combination, of a number of ways to inactivate or destroyendogenous germ cells, including by gamma irradiation, by chemicaltreatment, by means of infectious agents such as viruses, or byautoimmune depletion or by combinations thereof. This treatmentfacilitates the colonization of the recipient testis by the altereddonor cells. Animals that carry suitably modified sperm cells may beallowed to mate naturally, or alternatively their spermatozoa are usedfor insemination or in vitro fertilization.

In an exemplary embodiment, a transgenic animal may be produced by invitro infection of a single-cell embryo with a lentiviral vector. Seee.g., Lois et al., Science 295: 868-872 (2002).

Retroviral infection can also be used to introduce the transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A fourth type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

In general, progeny of transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the present inventionwill include exogenous genetic material encoding a contrast agent.Further, the sequence will preferably be attached to a regulatorysequence that allows the expression of the transgene. Contrast agentproduced in situ may be visualized by MRI.

8. MRI Methodologies

In general, contrast agents of the invention are designed for use in MRIdetection systems. In the most common implementation of MRI, oneobserves the hydrogen nucleus (proton) in molecules of mobile watercontained in subject materials. The subject material is placed in alarge static magnetic field. The field tends to align the magneticmoment associated with the hydrogen nuclei in water along the fielddirection. The nuclei are perturbed from equilibrium by pulsedradio-frequency (RF) radiation set at the Larmor frequency, which is acharacteristic frequency proportional to the magnetic field strengthwhere protons resonantly absorb energy. Upon removing the RF, the nucleiinduce a transient voltage in a receiver antenna; this transient voltageconstitutes the nuclear magnetic resonance (NMR) signal. Spatialinformation is encoded in both the frequency and/or phase of the NMRsignal by selective application of magnetic field gradients that aresuperimposed onto the large static field. The transient voltages aregenerally digitized, and then these signals may be processed by, forexample, using a computer to yield images.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES Example 1 NMR of K562 Cells Over-Expressing Ferritin: SimulatedTumor Studies

We describe data showing the feasibility of using of an over-expressionof intra-cellular metal-binding polypeptides as a potent MRI contrastagent. These initial results focus on ferritin in living human myeloidleukemia (K562) cells.

To investigate the sensitivity of ferritin in modulating the NMRproperties of K562 cells, we synthesized simulated “tumor” samples.These consisted of K562 cells that were stimulated to produce varyingamounts of excess intra-cellular ferritin in vitro. Cells were thensuspended in low-melting point agarose to form small pellets. Thespin-lattice relaxation rate (1/T₁) and the spin-spin relaxation rate(1/T₂) were measured in the pellets to quantify the impact of ferritin.(Modulation of these relaxation times give rise to image contrast inMRI.) In the same cells used for the samples, we assayed the totalferritin content using ELISA (Enzyme Linked Immuno-Sorbent Assay).

For the experiment, samples consisted of K562 cells that were stimulatedto over-express ferritin by a 16 hour incubation with varyingconcentrations of ferric ammonium citrate (FAC) in RPMI culture mediasupplemented with 2% fetal calf serum. After incubation, cells werewashed. For each FAC concentration, 10⁷ cells were counted for the NMRsample and 10⁶ cells we set aside for the ELISA assay (Alpha DiagnosticsInt. Inc., San Antonio, Tex.)). Cells used for the NMR samples werere-suspended in 50 μl of low melting point agarose in a small plastictube. The 1/T₁ and 1/T₂ measurements were performed at room temperatureusing a Bruker Minispec relaxometer (Bruker Instruments, Billerica,Mass.). Cells used for the ELISA were treated with lysis buffer and theconsistency of the total amount of released protein was confirmed usinga bicinchoninic acid protein quantitation assay (Pierce Inc., Rockford,Ill.). Ferritin concentration was calculated as an average over the cellpellet volume.

The correlation between the NMR changes and ferritin content is shown inFIG. 1. The results show substantial changes in the relaxation timeswith modest increases in ferritin expression over background; thesechanges are easily observed using MRI (below). These simulated tumorshave a cell density of 200 cells/nl.

Example 2 Toxicity Studies

The ferritin synthesis temporarily perturbs the cell's iron metabolism.Although the adverse effects of this on the cell's long-term health haveyet to be fully determined in vivo, indications from various in vitroexperiments have shown that ferritin overexpression is not harmful in avariety of cell lines, especially for transient expression. This wasconfirmed in our experiments in K562 cells described in Example 1 above.For each FAC concentration (and control), cells before and after theincubation period were counted 3-times using a hemocytometer and theresults were averaged. FIG. 2 shows the percent cells remaining afterthe 16 hour period of ferritin loading. In the simulated tumors,ferritin increases of greater than 10-times over baseline levels onlyresulted in a cell loss of order 20%. The ferritin increase required toprovide observable MRI contrast is only of order 2-4.

Example 3 MRI of Simulated Tumors

Ferritin over-expression in the simulated tumors is readily visualizedusing MRI. FIG. 3 shows a MRI image slice through three pellets used inthe NMR experiments. In this image, contrast is predominatelyT₂-weighted. In FIG. 3, (a) is the control, and (b)-(c) are the samplescontaining a ferritin increase of 2.7 and 4, respectively (see FIG. 1).Images were acquired simultaneously using a Bruker 7-Tesla MRI systemwith TE/TR=45/2000 ms, 128×128 image points, and a 1 mm-thick slice. Thepellet size was approximately 4 mm in diameter.

Example 4 MRI Studies of Cells Comprising Recombinant Ferritin

Both the light and heavy ferritin transgenes, denoted LF and HF,respectively, were introduced into variety of cell lines (e.g. K562 andRat 9L gliosarcoma) using lipid-based transfection methods and by usingviruses. The results were analyzed using ELISA, NMR, and MRI. Typicalresults are shown in FIGS. 4 and 5. Human light and heavy chain ferritincDNA having defective iron regulatory elements were used. Using standardmolecular biology techniques both transgenes were placed under thecontrol of the immediate early promoter of the CMV. The integrity of thetransgenes was confirmed by electrophoresis of DNA fragments followingdigestion with various restriction enzymes and by DNA sequencing.

Introduction of Ferritin via Transfection

9L cells (Fischer 344 rat gliosarcoma) were incubated in DMEMsupplemented with 10% fetal bovine serum (FBS), penicillin,streptomycin, and glutamine. Cells were plated in 24-well plates one daybefore transfection to achieve 60-80% confluence. The cells were rinsedwith serum-free DMEM and then covered with the same solution. A DNAmixture was prepared as follows. The reagent lipofectamine™ (Invitrogen,Carlsbad, Calif.) was combined with equal amounts of LF and HF DNA inserum-free DMEM. The reagent Plus™ (Invitrogen, Carlsbad, Calif.) wasadded to the DNA solution to increase transfection efficiency. The DNAmixture was added to the cells, and then incubated for 3 hours at 37°C., after which DMEM containing 10% FBS was added. Cells were collected48 or 96 hours post-transfection and counted. In addition, controlsamples were prepared by incubating 9L cells under identical conditionsas above, except that no DNA was added to the lipofectamine™—Plus™—DMEMmixture. Upon harvesting after 48 or 96 hours no significant differencesin cell numbers were observed between samples incubated with the DNAreporters and the control samples. Thus, there was no apparent toxicityassociated with the contrast proteins.

To assay the ferritin increase after transfection, 9L cells wereprepared as described above. The intracellular proteins were extractedusing the M-PER™ extraction Reagent (Pierce Biotechnology, MountainView, Calif.) and the ferritin content was assayed using an ELISA kit(Alpha diagnostics, San Antonio, Tex.). The results typically showed aferritin concentration ˜3 ng/ml in the transfected cells and anegligible (˜0.0 ng/ml) amount of human ferritin in the non-transfectedcells. (The 9L cell line is from rat, and the antibody used in the ELISAdetects only human ferritin with no cross-reactivity.)

The intracellular iron content was measured in transfected and controlcells to confirm an increased iron-uptake with transgene expression. Forthese experiments 20×10⁶ cells were plated and transfected using themethods described above. Control cells were also prepared as describedabove with no DNA added to the incubation solution. Cells were collected96 hours post transfection and counted. Using standard methods [2001Blood 97(9), 2863] cells were washed in PBS, and pellets were dissolvedin an acid solution and treated with a batophenan troline sulconatesolution. The light absorption of the solution was read at 535 nm usinga spectrophotometer and the iron concentration was calculated. Theresults indicate a factor of ˜1.5 increase in the net iron content ofthe transfected cells compared control.

Measurement of 1/T₂ in pellets of transfected cells was performed. Cells(20×10⁶) were transfected with the transgenes as described above. Cellswere collected 96 hours post-transfection, washed twice with PBS, andtransferred to a 0.2 ml micro-centrifuge tubes. Cells were againcentrifuged and the supernatant discarded. NMR measurements wereperformed on the pellets at 4° C. using a 20 MHz Bruker Minispec NMRanalyzer (Bruker Instruments, Billerica, Mass.). The results typicallyshow a factor of ˜15% increase in 1/T₂ in the transfected cells overcontrol.

Using the same cell pellets that were prepared for the above NMRexperiments, we confirmed that the 1/T₂ changes due to the expression ofthe contrast proteins provided satisfactory contrast in MR images. Themicro-centrifuge tubes containing the pellets were placed in an MRIapparatus and imaged using a standard T₂-weighted two-dimensionalFourier transform (2DFT) spin-echo pulse sequence. FIG. 4 displaystypical data and shows a high-resolution MRI slice through two pelletsacquired simultaneously; the left pellet is the control and the pelleton the right contains cells expressing the contrast proteins. Imagecontrast is clearly apparent between the two samples.

Introduction of Ferritin via a Viral Vector

Contrast proteins have also been introduced into cells via a viralvector. Infected cells were characterized using ELISA, NMR, and MRI. TheMRI data shows distinct contrast between cells infected with thecontrast proteins and uninfected (control) cells. For these experimentsthe LF and HF transgenes were each incorporated into separatereplication defective adenoviruses. These viruses were constructed usingthe commercially available Adeno-X™ expression system (Clontech, PaloAlto, Calif.) following the manufacture's instructions. The transgeneexpression was controlled using the CMV promoter. A HEK-293 cell linewas used for production of viral stocks. When the cytopathic effect wasevident in the HEK-293 cells due to viral production, cells werecollected, lysed, and the supernatants were collected. Thesesupernatants are adenovirus-rich and were used to infect mammalian cellsto demonstrate MRI contrasting effects. 9L cells were incubated in DMEMsupplemented with 10% FBS, penicillin, streptomycin, and glutamine.Cells (˜20×10⁶) were plated in 24-well plates one day before infectionto achieve 60-80% confluence. The cells were then rinsed with serum-freeDMEM and then covered with the same solution. Equal volumes of both theLF and HF adenovirus from each of the respective supernatants were addedto the 9L cells. The virus and cells were incubated in serum-free mediafor 0.5 hour, and then FBS was added to the DMEM to give 10% FBS. Aftera 48 hours incubation the cells were harvested, rinsed, and the effectsof the contrast genes were assayed. FIG. 5 shows typical MRI data of twopellets, infected and uninfected (control), 9L cells. These data wereacquired using a T₂-weighted 2DFT spin-echo sequence in a similar manneras the transfection experiments above. The left pellet is the controland the right pellet contains cells infected with LF and HF transgenes.Image contrast is clearly apparent between the two samples.

Example 5 Introduction of a NucleicAcid Encoding a Contrast Protein InVivo

This experiment is designed to demonstrate the delivery of contrastagent of the invention in vivo.

In this example, two tumor samples are transplanted onto a nude mouse.An HSV delivery is engineered to contain a nucleic acid constructcomprising the coding sequences for the human ferritins represented inSEQ ID Nos: 2 and 4. One tumor sample is injected with the HSV+ferritinvector, while the other tumor sample is injected with an “empty” HSVvector. The mouse is subjected to MRI, and the contrast between theHSV+ferritin sample and the “empty” HSV sample is compared.

Incorporation by Reference

All of the patents, publications and sequence database entries citedherein are hereby incorporated by reference. Also incorporated byreference are the following: Trinder et al., Int. J. Biochem. & CellBiol., 35: 292-296 (2003); Fleming et al., Proc. Natl. Acad. Sci. USA99: 10653-10658 (2002); and Fleming et al., Proc. Natl. Acad. Sci. USA97: 2214-2219 (2000).

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of generating an image of a subject material comprisingproviding a subject material comprising a plurality of cells wherein asubset of the cells express a recombinant nucleic acid encoding anMRI-detectable amount of ferritin; and detecting the cells by magneticresonance imaging caused by the expressed ferritin.
 2. The method ofclaim 1, wherein the cells comprising the measurable amount of ferritinare distinguishable from cells or other components of the material thatdo not comprise the measurable amount of ferritin.
 3. A method ofdetecting the expression of a recombinant nucleic acid encoding ferritincomprising providing a cell expressing a recombinant nucleic acidencoding ferritin; and detecting the cell by magnetic resonance imagingcaused by the expressed ferritin; wherein the detection of ferritin bymagnetic resonance imaging indicates that the nucleic acid encoding theferritin is and/or has been expressed.
 4. The method of claim 1 or 3,wherein the ferritin is selected from a group consisting of a proteincomprising SEQ ID NO: 2 and a protein comprising SEQ ID NO:
 4. 5. Themethod of claim 3, wherein the cell is part of a cell culture.
 6. Themethod of claim 3, wherein the cell is part of an in vitro tissue. 7.The method of claim 3, wherein the cell is part of a multicellularorganism.
 8. The method of claim 3, wherein the cell is part of amammal.
 9. The method of claim 3, wherein the cell is part of a plant.10. The method of claim 1 or 3, wherein the recombinant nucleic acid isoperably linked to a regulatory sequence.
 11. The method 10, wherein theregulatory sequence is active in situ.
 12. The method of claim 10,wherein the regulatory sequence is a constitutive regulatory sequence.13. The method of claim 10, wherein the regulatory sequence isexogenously regulated.